Previous work from this laboratory established that measurements of plasma metanephrines, O-methylated metabolites of catecholamines, provide a considerably superior diagnostic marker of pheochromocytoma than previous available tests. This follow-up study examines whether the superiority of plasma free metanephrines over other neurochemical tests for diagnosis of pheochromocytoma can be explained by production of metanephrines within tumors. Comparisons of the production of metanephrines in patients with pheochromocytoma with production from catecholamines released or infused into the circulation indicated that more than 93% of the high levels of circulating free metanephrines in patients with pheochromocytoma were derived from metabolism of catecholamines before and not after release into the circulation. Levels of metanephrines in pheochromocytomas over four orders of magnitude higher than plasma concentrations in the same patients before removal of tumors indicated production of metanephrines within tumors. Presence in pheochromocytoma tissue of catechol-O-methyltransferase (COMT), the enzyme responsible for conversion of catecholamines to metanephrines, was confirmed by enzyme assay, Western blot and immunohistochemistry. Immunohistochemistry revealed co-localization of COMT with tyrosine hydroxylase, the rate-limiting enzyme responsible for catecholamine synthesis. Enzyme assay and Western analysis indicated that membrane-bound and not soluble COMT was the prominent form of the enzyme and that there was more membrane-bound COMT in tumor and adrenal medullary tissue than in human liver. Since membrane-bound COMT has a much lower Km for catecholamines than soluble COMT the presence of the membrane-bound form of the enzyme in chromaffin tissue explains the large amounts of metanephrines produced by adrenal medullary and pheochromocytoma tissue. The data indicate that the elevated plasma levels of free metanephrines inpatients with pheochromocytoma are derived from catecholamines produced and metabolized within tumor tissue, this explaining the superiority of plasma metanephrines over other tests for diagnosis of pheochromocytoma; even when not actively secreting catecholamines the tumors are actively metabolizing catecholamines to metanephrines.